本文選題：CXCR1　切入點(diǎn)：腺病毒　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：CXCR1是只含有一條肽鏈的糖蛋白，為α亞族趨化性細(xì)胞因子受體。CXCR1可與IL-8特異性結(jié)合，介導(dǎo)細(xì)胞信號轉(zhuǎn)導(dǎo)的啟動，通過信號傳導(dǎo)過程，決定相應(yīng)配體趨化功能的實(shí)現(xiàn)，與細(xì)胞的活化、增殖、遷徙及刺激血管形成和釋放血管形成前體物質(zhì)等有關(guān)，因此CXCR1涉及到多種疾病的發(fā)生發(fā)展。為進(jìn)一步研究CXCR1在人體中各種疾病的發(fā)生發(fā)展中的作用，我們構(gòu)建CXCR1基因腺病毒表達(dá)載體，使得CXCR1基因在細(xì)胞內(nèi)得以表達(dá)。 目的：應(yīng)用AdEasy腺病毒載體構(gòu)建人CXCR1基因的重組腺病毒pAd-CXCR1。 方法：1.根據(jù)GenBank已收錄的CXCR1cDNA全長設(shè)計(jì)引物并送合成，采用PCR方法擴(kuò)增CXCR1基因。2.將目的基因的擴(kuò)增片段克隆至穿梭質(zhì)粒pAdTrace，構(gòu)建穿梭質(zhì)粒pAdTrace-CXCR1，并用KpnI和HindIII雙酶切進(jìn)行鑒定。3.PemI酶切pAdTrace-CXCR1使之線性化，用電轉(zhuǎn)化法將線性質(zhì)粒與腺病毒骨架質(zhì)粒pAdEasy-1轉(zhuǎn)入BJ5183細(xì)菌內(nèi)進(jìn)行同源重組，，構(gòu)建重組腺病毒質(zhì)粒pAd-CXCR1。4.重組腺病毒用酶切及PCR方法進(jìn)行鑒定。5.重組腺病毒質(zhì)粒用PacI線性化后轉(zhuǎn)染HEK-293細(xì)胞，進(jìn)行腺病毒的包裝，并在HEK-293細(xì)胞中進(jìn)行腺病毒擴(kuò)增。 結(jié)果：通過腺病毒構(gòu)建系統(tǒng)及酶切、連接、轉(zhuǎn)化等技術(shù)，成功的構(gòu)建了穿梭質(zhì)粒pAdTrace-CXCR1。將線性化的穿梭質(zhì)粒質(zhì)粒與腺病毒骨架質(zhì)粒pAdEasy-1于BJ5183細(xì)菌內(nèi)進(jìn)行同源重組，最終成功構(gòu)建重組腺病毒質(zhì)粒pAd-CXCR1。將pAd-CXCR1質(zhì)粒于HEK-293細(xì)胞內(nèi)擴(kuò)增，得到高滴度人CXCR1基因的腺病毒載體。 結(jié)論：成功構(gòu)建了人CXCR1基因的腺病毒載體，得到高滴度的腺病毒，為進(jìn)一步研究CXCR1蛋白的生物功能及其在臨床各種相關(guān)疾病中的作用奠定了基礎(chǔ)。
[Abstract]:CXCR1 is a glycoprotein containing only one peptide chain, is a subfamily of chemokine receptor.CXCR1 binds specifically to IL-8 mediated signal transduction, cells start, through the signal transduction process, determine the corresponding ligand to achieve function chemotaxis, cell activation and proliferation, angiogenesis, and release of precursor substances the formation of migration and stimulate the blood vessels, so CXCR1 is related to the occurrence and development of many diseases. For the role in the occurrence and development of the further study of CXCR1 in human diseases, we construct the expression vector of CXCR1 gene of adenovirus, the CXCR1 gene is expressed in cells.
Objective: to construct recombinant adenovirus pAd-CXCR1. of human CXCR1 gene by using AdEasy adenovirus vector
Methods: the full-length CXCR1cDNA primers were designed according to GenBank 1. have been included and sent to synthesis, amplification of the CXCR1 gene.2. gene amplification fragment was cloned into the shuttle plasmid pAdTrace by PCR method, and construct the shuttle plasmid pAdTrace-CXCR1, KpnI and HindIII were identified by double enzyme digestion of linear.3.PemI digestion to pAdTrace-CXCR1, the linear plasmid and adenovirus virus plasmid pAdEasy-1 for homologous recombination in bacteria into BJ5183 by electroporation method, recombinant adenovirus plasmid pAd-CXCR1.4. recombinant adenovirus by restriction enzyme digestion and PCR method for identification of.5. recombinant adenovirus plasmid transfected into HEK-293 cells by PacI after linearization of adenovirus, and adenovirus amplification in HEK-293 cells.
Results: through the construction of recombinant adenovirus system and enzyme digestion, connection, transformation technology, successfully constructed the shuttle plasmid pAdTrace-CXCR1. linearized shuttle plasmid plasmid and adenovirus backbone plasmid pAdEasy-1 for homologous recombination in bacteria BJ5183, finally successfully constructed the recombinant adenovirus plasmid pAd-CXCR1. pAd-CXCR1 plasmid in HEK-293 cell amplification, get gland high titer virus vector of human CXCR1 gene.
Conclusion: the adenovirus vector of human CXCR1 gene has been successfully constructed, and the high titer of adenovirus has been obtained, which laid a foundation for further studying the biological function of CXCR1 protein and its role in various clinical diseases.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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